When a functional group is introduced to a surface of a protein, specific protein residues are generally subjected to chemical modification. This chemical modification is simple, and many specific residues can be advantageously modified at one time. On the contrary, a favorable outcome is hard to attain in respect of the reproducibility of the modification site and/or the regulation of the number of modifications. Because of the progress in recent genetic engineering, it has become possible to substitute amino acid residues in a protein. Modification of a protein synthesis system has enabled the introduction of a desired non-natural amino acid having an amino acid skeleton to a protein and the synthesis of a functional-group-carrying protein with high reproducibility.
In the process of protein synthesis, an amino acid is first bound to the 3′ end of tRNA and then transferred to a ribosome where protein synthesis takes place. At the ribosome, codons are translated into amino acids. Use of tRNA comprising a non-natural amino acid bound thereto enables the incorporation of the non-natural amino acid into a protein. P. G. Schuss et al. and A. R. Chamberlain et al. Have reported a method that utilizes a termination codon to incorporate a non-natural amino acid into a protein by allocating UAG termination codon as a genetic code for a non-natural amino acid (Science, 244, p. 182, 1989, J. Am. Chem. Soc., 111, p. 8013, 1989). In this method that utilizes a termination codon, however, two or more kinds of non-natural amino acids cannot be introduced to a protein molecule, and the yield is disadvantageously low.
Sisido et al. Have developed a method that utilizes a four-basecodon in which a codon is comprised of 4 bases. A four-base codon is inserted into a mRNA at the site where a non-natural amino acid is intended to be introduced, an anticodon site of tRNA is substituted with a corresponding four-base, and a non-natural amino acid is then bound thereto. When protein synthesis is conducted using the modified mRNA and the anticodon, in the ribosome, a four-base codon-anticodon pair is formed at the site substituted with a four-base codon of mRNA, and the non-natural amino acid that was bound to tRNA is incorporated into an elongating peptide chain. In contrast, at other sites, triplet (3 bases) is decoded and translated as usual, thus non-natural amino acids are only introduced to the sites designated with a four-base codon in the final protein. Reference can be made to Hohsaka T. et al., J. Am. Chem. Soc., 118, 9778-9779, 1996 and Hohsaka T. et al., J. Am. Chem. Soc., 121, 34-40, 1999 (these documents are cited in this description by reference).
Further, Hirao et al. have developed a protein synthesis system utilizing an artificial base pair (a method that utilizes an artificial base codon) (Hirao, I. et al., Nature Biotech., 20, 177-182, 2002).
In these techniques, a non-natural amino acid can be bound to tRNA by chemical aminoacylation in which a dinucleotide at the 3′ end of tRNA is deleted, and a dinucleotide comprising a non-natural amino acid bound thereto is bound with the aid of an RNA ligase instead of the former dinucleotide. Further, more than one non-natural amino acid can be simultaneously introduced to a protein by the method that utilizes a four-base codon and by the method that utilizes an artificial base codon.
Introduction of a non-natural amino acid to a protein has enabled not only the analysis of protein structures and functions but the production of artificial proteins with some type of artificially-added functions. With the use of non-natural amino acids comprising a variety of label substances bound to the amino acid skeletons, therefore, labeled proteins into which the label substances have been incorporated can be obtained. With the method in accordance with each type of label substance, the labeled protein can be detected and/or purified directly or indirectly by means of enzymatic chemical techniques or enzymatic immunochemical techniques when, for example, the label substance is an enzymatic substrate or antigenic substance. This can be utilized in a wide variety of applications in the technical fields associated with medical sciences, pharmaceutical sciences, polymer chemistry, biochemistry, or the like.
It should be noted that a non-natural amino acid cannot be always introduced to a protein by merely binding to tRNA. A natural protein synthesis system has the admissibility of incorporating any of 20 types of naturally-occurring amino acids into a polypeptide chain, regardless of their types. Thus, a natural protein synthesis system is considered to have some degrees of admissibility for non-natural amino acids. However, an amino acid having a side chain with a bulky molecular structure such as 1-pyrenylalanine or ferrocenylalanine cannot be introduced to a protein through the natural protein synthesis system. In a natural protein synthesis system, the ribosome has two sites for incorporating tRNA. tRNA bound to a polypeptide is incorporated into one of them, and tRNA carrying an unreacted amino acid is incorporated into the other site. tRNA carrying a bulky amino acid, however, cannot be incorporated into the ribosome. Thus, tRNA carrying a bulky amino acid is deduced to be incapable of being incorporated into a protein.
Among label compounds, fluorescent substances have particularly high usefulness as labels for a protein. Further, a luminescent substance in the visible light range can be detected by a detector that is extensively and commonly used. Furthermore, a variety of highly sensitive detectors have already been developed and extensively used. These fluorescent substances are very useful as label compounds for labeling cells or the like since they are not affected by interferential actions caused by fluorescence emission in cells. When these fluorescent substances are used as label compounds for non-natural amino acids, however, it is difficult to introduce them to the proteins by a method that utilizes a protein synthesis system due to their large molecular weights. Compounds having a variety of functions, such as enzyme reactivity, antigenicity, protein binding property, or intermolecular interactivity, in addition to fluorescent and luminescent properties, are also useful as label compounds. The possibility of a label compound being introduced to a protein is disadvantageously limited by the method that utilizes a protein synthesis system due to its molecular weight. It is necessary to resolve this problem in the art.